The overall goals of our research are to characterize the mechanism by which consumption of alcoholic beverages increases the risk of APAP hepatotoxicity. CYP2E1 is considered responsible for alcohol-mediated increases in APAP hepatotoxicity. However, our work suggests that CYP3A also has a role. We have recently found that alcohols also induce CYP1A2, another form of CYP that produces the reactive metabolite of APAP. In this proposal, using knockout mice, we will investigate the roles of CYP2E1, CYP3A and CYP1A2 in alcohol-mediated APAP hepatotoxicity. The widely consumed chemicals caffeine and theophylline enhance the activity of CYP3A, and thus have the potential to augment alcohol-mediated APAP hepatotoxicity if consumed simultaneously with APAP. Therefore it is important to ascertain whether these methylxanthines are additional risk factors in alcohol-mediated APAP hepatotoxicity. In this proposal, we will also investigate the effects of caffeine and theophylline on alcohol-mediated APAP hepatotoxicity in rodents. HYPOTHESES We hypothesize that CYP3A can have a major role in the enhancement of APAP hepatotoxicity by alcohols. The actual contribution of a CYP in alcohol-mediated APAP hepatotoxicity will depend on the relative amount of that CYP as well as its activity in the liver at the time of exposure to APAP and the dose of APAP. The SPECIFIC AIMS of this proposal are: 1. To investigate the relative roles of CYPs 3A, 2E1 and 1A2 in APAP hepatotoxicity caused by pretreatment with ethanol and isopentanol, using CYP and reporter gene knockout mice. 2. To investigate the effect of methylxanthines, which increase CYP3A activity but decrease CYP1A2, on alcohol-mediated APAP hepatotoxicity in wild-type and CYP1A2 knockout mice.